Arch Gynecol Obstet
DOI 10.1007/s00404-014-3512-1
GYNECOLOGIC ONCOLOGY
The ratio of the estradiol metabolites 2-hydroxyestrone (2-OHE1)
and 16a-hydroxyestrone (16-OHE1) may predict breast
cancer risk in postmenopausal but not in premenopausal women:
two case–control studies
Xiangyan Ruan • Harald Seeger • Diethelm Wallwiener
Jens Huober • Alfred O. Mueck
Received: 28 August 2014 / Accepted: 7 October 2014
Ó Springer-Verlag Berlin Heidelberg 2014
Abstract
Purpose Two main estradiol metabolites have different
biological behavior with tumorigenic features of 16-OHE1
and antiproliferative characteristics of 2-OHE1. We
investigated the ratio of these estradiol metabolites in pre-
and postmenopausal patients with breast cancer (BC)
within two case–control studies.
Methods From 41 premenopausal patients with (cases)
and without (controls N = 211) BC and 207 postmeno-
pausal patients with and without BC (N = 206), urine
samples were collected. Urine samples were collected prior
to surgery and stored at -20 °C until measurement by
ELISA. The multiple linear regression test with two
interactions was performed to evaluate the influence of
different factors on the metabolic ratio.
Results In premenopausal patients, log ratio of 2-OHE1/
16-OHE1 was 0.25 (CI 0.20;0.29) and 0.21 (CI 0.11;0.31)
for controls and cases without significant difference. In
postmenopausal patients, log ratio was 0.22 (CI 0.17;0.26)
and 0.11 (CI 0.07;0.15) in controls and cases, respectively,
and was statistically significantly lower (p = 0.0002). Log
X. Ruan
A. O. Mueck
Department of Gynecological Endocrinology, Beijing Obstetrics
and Gynecology Hospital, Capital Medical University, Beijing,
China
H. Seeger
D. Wallwiener
A. O. Mueck (&)
Department of Endocrinology and Menopause, University
Women’s Hospital, Calwer Strasse 7, 72076 Tuebingen,
Germany
e-mail: alfred.mueck@med.uni-tuebingen.de
H. Seeger
e-mail: harald.seeger@med.uni-tuebingen.de
J. Huober
University Women’s Hospital, Ulm, Germany
•
ratio was significantly influenced by BMI, but only in
postmenopausal patients, an increased BMI resulted in a
significantly (p \ 0.042) decreased ratio.
Conclusions Our case control studies suggest that in
postmenopausal women a different metabolism of estro-
gens may play a role in the tumorigenesis of breast cancer.
This genetically determined metabolism could be influ-
enced by the exogenic factor BMI. In premenopausal
women different hormone levels at different time points of
the menstrual cycle may be an explanation that why we
could not find an influence of estrogen metabolism.
Keywords Estradiol metabolites
Breast cancer risk

Pre- and postmenopausal women
Introduction
Carcinogenesis is a multistep process in which estrogens
may play an important role. However, there are data that
not estradiol per se but several estradiol metabolites may
have carcinogenic potential [1]. In the human body estra-
diol is mainly metabolized by oxidative processes [2]. The
first stage is the transformation of estradiol into estrone by
oxidation in the C17 position, a process that is reversible.
The balance largely favors the formation of estrone, which
is characterized by the fact that estradiol is converted
rapidly into estrone, but the reverse reduction of estrone to
estradiol happens more slowly. Further metabolism from
estrone occurs irreversibly in two different ways, i.e., by
hydroxylations of estrone at the A-ring or at the D-ring.
Estrogen metabolism attracted attention because some
metabolites have been shown to elicit effects independent
of that of their parent substance 17ß-estradiol. Thus it
could be demonstrated that some metabolites have
123

proliferative, whereas other metabolites have antiprolifer-
ative properties [3]. Moreover, the D-ring metabolite 16a-
hydroxyestrone (16-OHE1) has been shown to elicit
genotoxic effects [4], whereas the A-ring metabolite
2-hydroxyestrone (2-OHE1) seems able to develop anti-
estrogenic actions by its conversion to the anti-estrogenic
compound 2- methoxyestradiol [5, 6]. In particular,
16-OHE1 has estrogenic activity which, based on the
increase of uterine weight of ovariectomized rats, is more
potent than that of estradiol [6]. In vitro studies revealed
two types of 16a-hydroxyestrone interactions at the estro-
gen receptor: a classical non-covalent bond with only
limited binding time, similar to that of estradiol; and a
covalent bond at the nuclear receptor, which was irre-
versible, probably inducing a long-term proliferative effect
on breast tissue [6].
Based on these preclinical data, it has been hypothesized
that patients metabolizing mainly via the D-ring-pathway
are at higher risk for developing breast cancer and that
screening for these metabolites possibly could identify
these patients. Several epidemiological studies have been
conducted so far to investigate a causal relationship
between the ratio of these metabolites and breast cancer
risk in pre- and postmenopausal women [7–15]. However,
differences in time of sample collection, choice of popu-
lation, low patient number may partly be responsible for
the inconsistent results found up to now. To see if a dif-
ferent metabolism is present in patients with breast cancer,
we investigated the ratio of 2-OHE1 to 16-OHE1 in these
patients and in patients with benign gynecologic diseases
comparing pre- and postmenopausal patients in two case–
control studies. Further we investigated if this genetically
determined ratio could be influenced by exogenic factors.
Patients and methods
Urine samples from 41 premenopausal women with breast
cancer diagnosis and 211 premenopausal women without
breast cancer as well as 206 non-breast cancer postmeno-
pausal patients and 207 postmenopausal women with pri-
mary diagnosis of breast cancer were collected at the
University Women’s Hospital of Tuebingen. The control
groups consisted of women with benign diseases of the
breast (fibroadenoma, mastopathy), and patients with
benign gynecological disorders. Patient history [menopause
state, smoking history, body mass index (BMI), hormone
therapy, alcohol intake, intake of drugs, former gyneco-
logical and non-gynecological diseases] was obtained by
using a questionnaire. Patients suffering from any kind of
hepatic disease, autoimmune diseases, nephropathy or
malignancies other than breast cancer were not allowed to
participate in this study. Patients with a history of breast
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Arch Gynecol Obstet
cancer and other gynecological cancers, excessive alcohol
consumption or medication influencing the hepatic meta-
bolic pathway were also excluded. The intake of oral con-
traceptives or hormone replacement therapy was only
documented for the last 4 weeks, whereby no information
on type, dosage and application form was recorded.
Urine samples were collected in the morning before
surgical treatment. To prevent degradation of the metabo-
lites, 1 mg ascorbic acid per 100 ml urine was added. The
samples were stored at -20 °C until measurement.
2-OHE1 and 16-OHE1 were measured by ELISA (ESTR-
AMET, IMMUNA CARE, Bethlehem, USA). The inter-
and intraassay variations were 14.3 and 10.5 % for
2-OHE1 and 15.9 and 9.9 % for 16-OHE1, respectively.
The specificity for the 2-OHE1 assay is 100 % for 2-OHE1
and 2OHE2, 68 % for estetrol, and\1 % for other relevant
estradiol metabolites. The specificity for the 16-OHE1
assay is 100 % for 16-OHE1 and \1 % for other relevant
estradiol metabolites. Absolute values expressed in ug
steroid hormone/mg creatinine were compared after loga-
rithmic transformation (log ratio 2-OHE1 to 16-OHE1) by
t test. The multiple linear regression test with two inter-
actions was performed to evaluate the influence of different
anamnestic factors on the metabolic ratio.
Results
The basic characteristics for both premenopausal controls
and cases and postmenopausal controls and cases are
shown in Table 1. Except for an imbalance of smokers in
the premenopausal groups, there were no significant dif-
ferences between controls and cases. In Table 2, the benign
diseases of the control group are listed. In Table 3, the
diagnostic and histological data of the breast tumors are
given.
Absolute mean values for 2-OHE1 and 16-OHE1 in pre-
and postmenopausal patients expressed as lg/mg creatinine
are stated in Table 4. In premenopausal women in the
control group values of 23.09 ± 23.7 and 12.2 ± 12.2 were
found for 2-OHE1 and 16-OHE1, respectively. The corre-
sponding figures for the cases were not significantly lower
than those for the controls with values of 18.5 ± 14.7 and
10.0 ± 7.3, respectively. In contrast in the postmenopausal
women the absolute values of both metabolites dif-
fered significantly. In the control population values of
19.5 ± 26.1 and 11.9 ± 18.3 were found for 2-OHE1 and
16-OHE1, respectively. The corresponding values for the
cases were significantly lower than those for the controls
with values of 10.2 ± 14.1 and 7.9 ± 11.3, respectively.
Further we compared the absolute values after loga-
rithmic transformation (log ratio 2-OHE1 to 16-OHE1)
because there is a high variation in the urinary excretion of
Arch Gynecol Obstet

Table 1 Basic characteristics of pre- and postmenopausal women Table 3 Diagnostic and histo- N
with and without breast cancer (mean ± SD) logical data of the tumors
Diagnosis breast cancer
Controls Cases p value
N = 211 N = 41 Inv. Ductal 155
Inv. Lobular 57
Premenopausal
DCIS 25
Age 40.5 ± 9.0 44.0 ± 6.3 ns
Others 11
Height 165.5 ± 6.1 165.8 ± 5.2 ns
Histological classification
Weight 67.7 ± 13.2 64.4 ± 14.1 ns
T1 134
BMI 24.7 ± 4.8 23.4 ± 4.6 ns
T2 64
Current smoker 77 28 0.01
T3 5
Alcohol 123 17 ns
T4 2
Controls Cases p value N pos 73
N = 206 N = 207
Her2/neu pos 41
Postmenopausal
Age 59.5 ± 8.1 62.7 ± 8.5 ns
Height 164.6 ± 5.7 163.7 ± 6.1 ns
Weight 70.7 ± 11.7 69.9 ± 11.6 ns Table 4 Absolute values (lg/mg creatinine) of the estradiol metab-
BMI 26.1 ± 4.4 26.1 ± 4.0 ns olites 2-hydroxyestrone (2-OHE1) and 16a-hydroxyestrone (16-
Current smoker 93 77 ns OHE1) in pre- and postmenopausal women with and without breast
cancer (mean ± SD, * differences between groups)
Alcohol 101 95 ns
Hormone intake 110 61 ns Controls Cases p value
N = 211 N = 41

Premenopausal
Table 2 Benign breast diseases 2-OHE1 23.09 ± 23.7 18.5 ± 14.7 ns
Benign breast diseases
and benign gynecological 16-OHE1 12.2 ± 12.2 10.0 ± 7.3 ns
diseases in the control group Mastopathia 46
Fibroadenoma 22 Controls Cases p value
Other 8 N = 206 N = 207
Benign gynaecologic disorders Postmenopausal
Myoma 113 2-OHE1 19.5 ± 26.1 10.2 ± 14.1 \0.0001*
Endometriosis 23 16-OHE1 11.9 ± 18.3 7.9 ± 11.3 0.0064*
Endometrial hyperplasia 6
Ovarian cysts and 38
fibroadenoma
Polyps 28 0,35
Log ratio 2-OHE1 / 16-OHE1

Incontinence 34 0,3
CIN 1–3 13 0,25

0,2

0,15
estradiol metabolites. In premenopausal patients, the log 0,1
ratio was 0.25 (CI 0.20;0.29) and 0.21 (CI 0.11;0.31),
0,05
respectively for controls and cases without significant dif-
ference, as shown in Fig. 1. In postmenopausal patients 0
Controls Cases
(Fig. 2), we found a statistical significant higher ratio of
0.22 (CI 0.17;0.26) in controls as compared to 0.11 (CI Fig. 1 Log ratio of 2-hydroxyestrone (2-OHE1) to 16a-hydroxye-
0.07;0.15) in cases (p = 0.0002) suggesting a relative strone (16-OHE1) in premenopausal women without (controls
N = 211) and with (cases N = 41) breast cancer. (means, 95 % CI)
outweigh of the tumorigenic metabolite 16-OHE1 in breast
cancer patients.
Multiple linear regression analysis was done to evaluate premenopausal patients none of these factors significantly
the influence of hormone receptor expression, BMI, influenced the metabolic ratio as shown in Table 5. Con-
smoking and hormone intake on the metabolic ratio. In versely in the postmenopausal patients an increased BMI

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0,35 associated with a low 2/16 ratio. Thus in the postmeno-

Log ratio 2-OHE1 /16-OHE1

0,3 pause, BMI appears to have a negative influence on

0,25
estradiol metabolites. BMI is an independent, important
breast cancer risk factor and the negative influence on
0,2
estradiol metabolite ratio might contribute among other
0,15 ** effects of BMI such as on E1/E2 synthesis, SHBG syn-
0,1 thesis and IGF-1 production [16].
0,05 In our study smoking had no influence on 2-OHE1/16-
0
OHE1 ratio. However, earlier studies suggest an effect of
Controls Cases smoking on estradiol metabolism [17, 18], especially
increasing 2-hydroxylation of the parent estrogen, estra-
Fig. 2 Log ratio of 2-hydroxyestrone (2-OHE1) to 16a-hydroxye-
diol. In a recent publication smoking has been attributed to
strone (16-OHE1) in postmenopausal women without (controls
N = 206) and with (cases N = 207) breast cancer. (means, 95 % an increased 2- and 4-hydroxylation and a decreased
CI; **p \ 0.01 vs. controls) 16-hydroxylation measured by liquid chromatography [19].
In our study also no influence on estradiol metabolism
was found for hormone intake.
Table 5 Factors influencing the response variable case–control. Exogenous hormone therapy has been shown to increase
Results of multiple linear regression
overall metabolite concentrations in the case of oral
Factor Estimate SD p value application as compared to transdermal one [2], but no
Premenopausal alteration was found for the 2/16 ratio [2]. Combination of
BMI -0.020 0.010 0.057
smoking with exogenous hormone intake has been shown
Smoking 0.164 0.053 0.081
to influence estradiol metabolism. For example, in a direct
comparison with female non-smokers, it was demonstrated
Hormone intake 0.012 0.067 0.806
that women smokers under treatment with oral estradiol
Estrogen receptor -0.001 0.155 0.516
(2 mg/day) produce significantly more 4-hydroxyestradiol,
Progesterone receptor -0.064 0.079 0.282
which (via quinone formation) can have DNA-toxic
Estrogen/Prog-Rec -0.075 0.188 0.694
effects, and at the same time they produced smaller
Postmenopausal
amounts of 2-methoxyestradiol, which has anticarcino-
BMI -0.016 0.007 0.015
genic effects [20].
Smoking 0.043 0.033 0.098
Various observational trials have demonstrated that the
Hormone intake -0.003 0.028 0.783
ratio of 2- to 16a-hydroxyestrone is decreased in women
Estrogen receptor 0.002 0.058 0.960
with breast cancer. However, existing studies have had
Progesterone receptor 0.032 0.03 0.406
different designs, have been conducted in different popu-
Estrogen/prog-Rec 0.009 0.075 0.904
lations, and some have extremely small sample sizes. Thus
their results are inconsistent, with some showing a strong
leads to a highly significant (p = 0.030) lower ratio of inverse association of increasing levels of the 2-OHE1/16-
2-OHE1/16-OHE1. The other tested factors were without OHE1 ratio with breast cancer [7, 8], some showing a
influence on the ratio. modest association [9–12], and still others showing no
association [13, 14]. A study conducted in China [10]
found an inverse association of the 2/16 ratio with breast
Discussion cancer using urine samples collected before surgery, but a
positive association using postsurgery samples. In a recent
In our case–control studies, the log ratio of 2-hydroxye- large population-based case–control study, an inverse
strone to 16a-hydroxyestrone was investigated in pre- and association of breast cancer risk with 2/16 ratio was found
postmenopausal women with and without breast cancer. to be mostly consistent for premenopausal women with
Only for postmenopausal women, a significant difference invasive breast cancer [13].
of the ratio was found; it was lower in the women having Recent trials on the 2/16 ratio and breast cancer risk used
breast cancer. more sophisticated detection methods as there are liquid
The results of the linear regression in terms of diverse chromatography–tandem mass spectrometry. In a case–
factors that might influence the ratio, such as BMI, control study [21], 2- and 4-hydroxyestradiol were increased
smoking, hormone intake and hormone receptor expres- in cancer patients, whereas 16-OHE1 and 2-methoxyestra-
sion, revealed that only BMI had a significant influence and diol were reduced in the breast cancer group. In postmen-
only in the postmenopausal group. Here, a high BMI was opausal women serum estrogen concentrations were

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associated with breast cancer risk, especially more
2-hydroxylation lowered the risk, whereas less methylation
of 4-hydroxyestrogens increased breast cancer risk [22].
More extensive 2-hydroxylation was also associated with a
lower breast cancer risk in serum samples of a nested case–
control study [23]. In contrast in premenopausal women, no
association was found for urinary estrogen metabolites and
breast cancer risk [24]. However, in premenopausal women
the 2-OHE1/16-OHE1 ratio was significantly increased after
exercise intervention and, thus, may reduce breast cancer
risk that is observed in women with physical activity [25].
There are several limitations to our analysis: We have
only measured estradiol metabolites in urine. It has been
demonstrated that intracellular estrogen concentrations can
be up to ten times higher than serum concentrations [26],
but it is unknown in as far urinary excretion may be cor-
related with the intracellular activities. Research on intra-
cellular levels of estradiol metabolites have so far mainly
focused on the 4-hydroxylated metabolites. Thus elevated
4-hydroxylase enzyme activity has been found in human
breast cancer specimens, and 4-hydroxyestradiol as well as
the quinones was found in high concentrations in human
breast cancer tissue [27, 28].
In addition we only measured estrogen metabolites in
a single urine sample, which may not accurately reflect
long-term exposure. The sample size in the group with
premenopausal women having breast cancer was rather
small, thus conclusions drawn for this group of patients
might be handled cautiously. Furthermore, we cannot
discriminate between luteal and follicular urine samples
for the premenopausal women and so far it is unknown,
if a possible different association between follicular and
luteal urine and estradiol metabolism pattern may exist.
Acknowledgments This work was supported by grants to Xiangyan
Ruan for scholarships in Clinical Pharmacology at the University of
Tuebingen, Germany, by the following funding projects: Beijing
Municipality Health Technology High-level Talent (No. 2009-3-52)
and National Natural Science Foundation (No. 81172518) and Project
of discipline leader, Beijing Obstetrics and Gynecology Hospital,
Capital Medical University, Beijing maternal and Child Health Care
Hospital (2013-1).
Conflict of interest The authors report no conflict of interest. The
authors alone are responsible for the content and writing of this paper.
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